Notch signaling enhances proinflammatory functions of alloreactive T cells and suppresses the expansion of alloantigen-specific regulatory T cells after transplantation. BALB/c recipients were lethally irradiated (800 cGy) and transplanted with B10.D2-Thy1.2 TCD BM (10⁷ cells) plus 12 × 10⁶ B10.D2-Thy1.1 WT or B10.D2-DNMAML splenocytes (labeled with eFluor450 to allow for tracking of cell division). Tissues were retrieved for flow cytometric analysis of alloantigen-specific Vβ3⁺ T-cell populations. (A) CD25 expression in CD4⁺Vβ3⁺ T cells at days 2 and 6 after transplantation. *P < .01 (2-tailed unpaired Student t test). (B) eFluor450 dilution at days 2 and 6 posttransplant showing preserved early proliferation of DNMAML as compared with WT CD4⁺Vβ3⁺ T cells. (C-D) Flow cytometric analysis of cytokine production by WT vs Notch-deprived DNMAML CD4⁺Vβ3⁺ T cells isolated from spleen or liver at day 6 after allo-HCT (ex vivo anti-CD3/CD28 restimulation before staining for intracellular cytokines). Representative contour plots are shown in panel C and cumulative data in panel D. Numbers indicate the percentage of events falling within indicated rectangular gates. *P < .01 (2-tailed unpaired Student t test). (E) Notch inhibition in Vβ3⁺CD4⁺ T cells alters transcription of Notch targets but does not affect Th-lineage polarization. RNA was isolated; a complementary DNA library was generated from sort-purified Vβ3⁺CD4⁺ T cells on day 6 after allo-HCT and then subjected to quantitative reverse-transcription PCR. While Notch targets were depressed (Dtx1), canonical Th1 (Tbx21) and Th2 (Gata3) transcription factors remained unaffected. (F-G) Notch inhibition enhances expansion of Vβ3⁺CD4⁺FoxP3⁺ Tregs after allo-HCT. Data show expression of FoxP3 in Vβ3⁺CD4⁺ T cells 6 days after allo-HCT (F) and total Vβ3⁺ Tregs at that time point (G). *P < .01 (2-tailed unpaired Student t test). Data in panels A-F are representative of at least 2 experiments with 10 animals per cohort.